Mycobacterium tuberculosis(Mycobacterium Bovis) antibody ELISA test kit

Catalog No. LSY-30046

1. Introduction

The Mycobacterium tuberculosis(Mycobacterium Bovis) antibody ELISA kit is used to test Mycobacterium tuberculosis antibody in bovine serum. It is used to evaluate the serologic assistant diagnosis of tuberculosis caused by Mycobacterium bovis.

This kit use indirect ELISA method, Mycobacterium tuberculosis antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is Mycobacterium tuberculosis specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-Mycobacterium tuberculosis antibody, it will combine with the complex of antibody and pre-coated antigen on plate; discard the uncombined enzyme conjugate with washing; Add TMB substrate in micro-wells, blue product formed enzymatically, use ELISA reader at 450nm wavelength to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

2. Reagents and contents

3. Materialrequired notprovided

1) Micropipette: 0.5µl-10µl, 10ul-100ul, 100ul-1000ul.

2) Disposable pipette tips.

3) Graduate: 500ml.

4) Microplate Reader: 96 wells.

5) Distilled water or deionized water.

6) Microplate Washer

4. Sample preparation

Take animal whole blood, get serum by using regular method, the serum should bright and no hemolysis

5. Washing buffer preparation

Return 10X Concentrated washing buffer into room temperature before use, if  there is salt crystals, shake to make it dissolved, then dilute it at 10 times with distilled water or deionized water. The diluted washing buffer can store at 4℃for about 1 week.

6. Sample dilution

Dilute the serum sample with Sample diluent at 1:99(for example: 495ul sample diluent + 5ul serum sample)

Notice: Negative control and Positive control do not need dilute. Exchange tip after taking sample every time, record the position of the sample on plate accurately. Shake the sample evenly before adding it.

7. Notes

1) Return all reagents into room temperature before use, shake it evenly before use, and store back to 2-8℃after usage.

2) Do not mix use reagents from different kits and different lot no., prevent the reagents been polluted when using.

3) Substrate and stop solution may have irritation to skin and eyes, be careful to use.

4) Do not expose Substrate to strong light and avoid contact with the oxidant.

5) Tuberculosis-Ag coated plates should be sealed and moisture-proof. Put back unused MicroWell plate into dry foil bag and sealed at 2~8 ℃.

6) All wastes should be treated well to avoid pollution before discarding.

7) Strict compliance with the operating instructions can get the best results. Pipetting operation, timing, and washing of the whole process must be precise.

8) Serum dilution plate is disposable, do not repeat use. The maximum capacity of the serum dilution plate was 300ul/well.

8. Test procedure

1) Take Tuberculosis-Ag Coated plates (open and take the quantity required for use according to sample quantity), add diluted serum into test wells, 100ul/well; meanwhile set 2 wells for positive control and 1 well for negative control, add negative control and positive control into its wells accordingly, 100ul/well(do not spill);

2) Cover it with Adhesive Foil, incubate at 37℃for30 minutes;

3) Open the adhesive foil, discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, discard the liquid, repeat the above step for 4-6 times, at last flap to dry with the absorbent paper;

4) Adding Enzyme Conjugate,100ul/well, shake gentle to mix it evenly, cover it with Adhesive Foil, incubate at 37℃for30 minutes;

5) Open the adhesive foil, discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, discard the liquid, repeat the above step for 4-6 times, at last flap to dry with the absorbent paper;

6) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat 37 ℃in darkfor10 minutes;

7) Add stop solution, 50ul/well to stop the reaction, measure the result in 10 minutes.

9. Results judgement

Read the OD value at 450nm (630nm as reference).

For the assay to be valid:

OD value of negative control(N) < 0.2, meanwhile OD value of positive control (P) > 0.4

Calculate method:

S/P value= Sample OD value/ Positive control OD average value

Resultsinterpretation

S/P value < 0.3:  Negative

S/P value)≥ 0.3: Positive

10. Storage and expire date

Store at 2~8℃ in dark, no frozen, expiry date: 12 months.